Type A ?-aminobutyric receptors (GABAAR) are Cl--preferring ligand gated ion channels that mediate fast phasic synaptic inhibition in the adult brain and are are drug targets for barbiturates, benzodiazepines, intravenous anesthetics, and neurosteroids. Consistent with their essential role in regulating neuronal excitability, modification in GABAAR activity contribute to anxiety, autism, depression, epilepsy, substance abuse and schizophrenia. The receptors are heteropentamers that can be assembled from ?(1-6), ?(1-3), ?(1-3), ?, ?, ? and ? subunits. Surprisingly, to date our comprehension of the molecular structure of native GABAARs subtypes, and how these proteins are targeted to inhibitory synapses is rudimentary. However, these issues are of fundamental importance given the critical role GABAARs play in determining neuronal excitability, in neuropsychiatric diseases and as drug targets. To address these issues, we will determine the structure of the principal GABAAR subtypes assembled from ?1 and ?2 subunits in the brain. We will then assess which proteins co-purify with these receptor subtypes. The roles that the most abundant ?receptor-associated proteins? play in the synaptic targeting of distinct GABAAR subtypes will then be examined. Our studies will be facilitated by the novel mouse lines we have developed in which the N-terminus of the ?1 and ?2 subunits have been modified with distinct fluorescent reporters; pHlourin/9E10-?2 (pH?2) and mKate/FLAG-?1 (mK?1). These additions are functionally silent but allow sequential affinity purification of individual GABAAR subtypes, and analysis of their structure using blue-native polyacrylamide gel electrophoresis (BN-PAGE) followed by liquid chromatography coupled mass spectroscopy (LC-MS/MS). Preliminary experiments using these new tools have allowed us to formulate a central hypothesis that will be tested here; ?2- and ?1-subunits are assembled into two distinct GABAAR subtypes; ??2/?1? containing equimolar amounts of both ? subunits (?2?1??2), and ??1/?1? which does not contain an ?2 subunit (?1??2). These subtypes are targeted to distinct subsets of synapses by spectrins, which are intimately associated with these GABAAR subtypes. Our experiments will focus on the following specific aims: Aim 1. To test the hypothesis that neurons assemble GABAAR subtypes containing ?2?1??2 and ?1??2 subunits. Aim 2. To test the hypothesis that the ?2/?1 and ?1/?1 GABAAR subtypes are associated with distinct spectrins. Aim 3. To test the hypothesis that spectrins facilitate the synapse-specific targeting of GABAARs. Collectively, our study will provide novel insights into the structure of native GABAARs and the processes that regulate their accumulation at inhibitory synapses, information that may lead to improved understanding of, and treatments for epilepsy and other neuropsychiatric disorders.